Solid Phase Derivatizations in HPLC: Borohydride/Silica Reductions for Carbonyl Compounds

Abstract

Sodium borohydride adsorbed onto silica gel has now been utilized for on-line, pre-and post-analytical column chemical derivatizations via reductions of various organic carbonyl compounds. These on-line reactions have been performed using normal phase HPLC conditions, involving conventional silica gel packings, organic mobile phases, and commercially available HPLC equipment and instrumentation. This approach for on-line HPLC derivatizations has been evaluated for a large number of organic carbonyl compounds, at a variety of temperatures for aldehydes and ketones. The overall rates of such carbonyl reductions via sodium borohydride/silica are sufficiently different as a function of temperature of the reaction to permit for compound/class identifications. Analyte identification can be both qualitative and quantitative, even wherein an analyte co-elutes with a non-carbonyl compound. In-house prepared borohydride/silica gel reactors can be characterized via standard iodine titration procedures along with inductively coupled plasma (ICP) elemental boron analysis. These normal phase derivatization approaches have been applied to certain standard vitamins, and to cinnamaldehyde found in a commercial spice product. Such approaches to on-line HPLC derivatizations     

Silica Based 3,5-Dinitrobenzoyl (Dnb) Reagent for Off-Line Derivatization of Amine Nucleophiles in HPLC

Abstract

We describe here a new silica based derivatization reagent, containing the 3,5-dinitrobenzoyl tag, for solid phase derivatization of amines. It can be used for the off-line derivatization of primary and secondary amines. the amide derivatives can be easily detected under conventional UV detection modes. the entire synthetic method, structural characterization, and optimization of derivatization conditions of this solid phase derivatization reagent are described, Also, the reagent was tested in the on-line, pre-column derivatization mode for reversed phase HPLC, as well as for histamine analysis in fish samples     

New polymeric benzotriazole reagents for off-line derivatizations of amines and polyamines in HPLC

New polymeric reagents are synthesized, based on a polystyrene-bound benzotriazole containing an o-acetylsalicyl or 9-fluorenyl labelling moiety. This is used in an off-line mode, prior to high performance liquid chromatography (HPLC), for derivatizations of trace primary and secondary amines, polyamines, and related compounds in connection with HPLC. Standards are prepared, characterized by physical and spectral properties, and then used as external standards to determine percent derivatizations. The polymeric reagents are characterized by elemental analyses and loading determinations. The feasibility and applicability of this reagent for derivatization of nucleophiles is confirmed with a number of amines under optimized conditions. The activated labelling moiety, bonded to the polymeric support, makes the derivatization reactions extremely rapid and efficient under mild reaction conditions. This alone provides significant advantages over the analogous solution derivatizations for the same amines. A comparison of solution and solid phase derivatization reactions is reported. The limits of detection are 1 to 2 pmol for polyamines, such as cadaverine, putrescine, and 1,7-diaminoheptane, using the benzotriazole fluorenyl reagent followed by fluorescence detection.     

A mixed-bed, multi-derivatization approach using polymeric reagents for derivatizations of amines in high performance liquid chromatographic detection

Immobilized, polymeric reagents containing covalently attached tagging groups have been synthesized and reacted individually, off-line or on-line, pre-column in high performance liquid chromatographic (HPLC) detection. These reagents have also been combined into a single, mixed-bed reactor, useful for simultaneously preparing several derivatives from a single analyte, all at the same time. Each derivative possesses different chromatographic and detection properties, dependent on the nature of the original polymeric reagent containing the immobilized, tagging species. These particular reagents were designed to impart Ultraviolet/fluorescence, Ultraviolet/electrochemistry (oxidative/reductive or oxidative-hv-electrochemistry) to the final derivatives. Variations in the amounts/ratios of polymeric reagents contained in a single mixed-bed reactor will lead to varying ratios of the final derivatives. These can be predicted knowing the approximate reactivity of each polymeric reagent, percent derivatizations, and overall rates for each reagent towards a given substrate. In this first example of mixed-bed, polymeric reagents for improved derivatization approaches in chromatography, emphasis has been placed on simple amines or amine-like analytes. Multiple derivatives can be effectively used to improve the identification of an unknown analyte in a complex sample matrix, as well as to improve the detectability of that analyte. As one real world application, amphetamine in human urine was quantitated via on-line derivatizations with a mixed-bed reactor. With the least sampling work-up, the resulting sample solutions were directly injected into the on-line derivatization HPLC system for quantitation. The method was validated by single blind spiking experiments. The precision and accuracy were acceptable.     

Derivatization and Post-Column Reactions for Improved Detection in Liquid Chromatography/Electrochemistry

Abstract

A summary Is provided of most of Che reported derlvatizations that have been used for improved analyte detection in liquid chromatography with electrochemical detection (LCEC). These approaches include pre-column derivatizations and postcolumns chemical, photochemical or enzymatic reactions for oxidative EC detection. This review covers the literature up to early 1985, and includes information gathered from books, technical articles, previous reviews and scientific journal publications. Specific reagents, methods and instrumentation are described for those classes of compounds studied by derlvatization-LCEC, and suggestions for future experiments are included, where applicable. It is concluded that the future will likely Include the development of a great number of derivatizations which may be used in conjunction with LCEC for trace analysis.     

Liquid Column Chromatography with Chemical Derivatizations after Separation. [New analytical methods (15)]

Modern liquid column chromatography (high-pressure liquid chromatography, HPLC) has evolved in the last few years into a highly efficient and versatile separation technique. The selectivity of an analytical process that depends upon a previous separation step can in many cases be increased considerably by chemical derivatizations after the separation. In addition, lower detection limits can be achieved in this way than in detection without derivatization. The physicochemical principles of these combined processes involving chromatographic separation and chemical derivatization prior to detection (coupling of HPLC and a reaction detector) are presented and discussed. The state of development is outlined, with a survey of the more important applications so far described in the literature.     

MMNTP-a new tailor-made modular derivatization agent for the selective determination of isocyanates and diisocyanates

The synthesis of a new tailor-made derivatization agent for the selective determination of (di)isocyanates is presented. Starting from cyanuric chloride, the reagent 4-methoxy-6-(4-methoxy-1-naphthyl)-1,3,5-triazine-2-(1-piperazine)(MMNTP) is synthesized by subsequent substitution of the three chlorine atoms. This new derivatization agent and the five urea derivatives of phenylisocyanate (PI), hexamethylene-diisocyanate (HDI), toluene-2,4-diisocyanate (2,4-TDI), toluene-2,6-diisocyanate (2,6-TDI) and methylenebisphenyl-4,4-diisocyanate (MDI) show good spectroscopic properties with small compound-to-compound variabilities (RSD([epsilon])= 5.3 %, RSD(relative fluorescence)= 9.4 %). Therefore, using UV detection, a single calibration is needed for the quantification of all diisocyanates and isocyanates respectively. For separation and analysis a HPLC method with a RP column and a binary gradient is presented. All derivatives are separated and show low limits of detection. In addition to the good spectroscopic properties and low limits of detection, good reactivity for the derivatizations at room temperature is observed. The aromatic diisocyanates can be measured immediately whereas aliphatic diisocyanates need 2 h incubation. These advantages make MMNTP a powerful and versatile derivatization agent for (di)isocyanates which is demonstrated by a real sample with solid phase sampling, where the reagent is coated on a sorbent.     

Handling of Environmental and Biological Samples via Pre-Column Technologies

Abstract

Sample handling is still a weak point in chromatography and in analytical chemisty in general. One consideration is the automation potential of new procedures. Solid-liquid extraction techniques in combination with pre-column technology are particularly promising in this regards. The construction and geometry of pre-columns both for conventional and narrow-bore HPLC are of major importance, since band broadening should be kept at a minimum for an optimal functioning of the analytical system. The various operations that can be carried out with such a pre-column are trace-enrichment, clean-up of the sample which depends on the type of adsorbents used in the precolumn, i.e., polar or apolar materials, ion exchangers or metal covered surfaces, etc., protection of the analytical column, field sampling and storage of samples and as a substrate for on-column chemical derivatizations. These various operations are demonstrated with practical examples from the fields of environmental and biological analysis. The selectivity can be further enhanced by coupling precolumn technology with selective detection modes such as diode array UV, electrochemical or fluorescence detection. This enables the construction of optimal and integrated analysis sytems which are fully automated and microprocessor controlled. They can also be made compatible with miniaturized LC-technology.     

Solid-phase reagent containing the 3,5-dinitrophenyl tag for the improved derivatization of chiral and achiral amines, amino alcohols and amino acids in high-performance liquid chromatography with ultraviolet detection

A solid-phase reaction technique is described for improved derivatization of aliphatic amines, amino alcohols and amino acids. A polymeric activated ester is used for the immobilization of the 3,5-dinitrobenzoyl group, which can then be used for derivatizations of strong or weak nucleophiles, while avoiding solution-phase derivatization conditions. The reagent is easily prepared and can be regenerated after use to attain its original reactivity. The resulting chromatograms are free of system peaks due to excess derivatizing reagent, and sample handling is kept to a minimum. The reagent can be used in conjunction with both reversed- and normal-phase chromatography and can be used for off-line gas chromatographic or high-performance liquid chromatographic (HPLC) derivatizations. In addition, the reagent can be used on-line for derivatization in HPLC. Since the labelling reagent is a strong pi-acid, chiral substrates can be derivatized and separated on a Pirkle-type pi-donor column. The confirmation and quantitation of amphetamine in urine was accomplished using a polymer containing two labelling moieties, p-nitrobenzoyl and 3,5-dinitrobenzoyl. The derivatization and separation of chiral and achiral amines, amino alcohols and amino acids is described.     

A method for the determination of histamine in wine by HPLC with precolumn derivatization with phenylisothiocyanate

Summary A method for determining histamine in wine by precolumn derivatization with PITC (phenylisothiocyanate) with reversed-phase HPLC and UV detection is reported. Histamine can be determined together with the 24 amino acids within 40 min, or separately in a shorter time (less than 4 min) if a prior solid phase extraction clean-up is used.     

高效液相色谱法测定茶叶中的茶氨酸

建立了未衍生化高效液相色谱法(HPLC)测定茶叶中茶氨酸含量的方法。采用的色谱条件为:C 18 色谱柱,以0.05%(体积分数)三氟乙酸水溶液为流动相,流速1 mL/min ,进样量10 μL,检测波长203 nm。茶氨酸质量浓度在0.02～1 g/L 内,其浓度与峰面积呈良好的线性关系,最低检出限为1.75 ng(S/N=3),回收率为97.2%,相对标准偏差(RSD)为1.7%。同时以高效液相色谱-电喷雾离子化质谱对所分离的茶氨酸进行了纯度鉴定。方法具有精确、灵敏、流动相组成简单等特点。     

Derivatization procedures and their analytical performances for HPLC determination in bioanalysis

Derivatization, or chemical structure modification, is often used in bioanalysis performed by liquid chromatography technique in order to enhance detectability or to improve the chromatographic performance for the target analytes. The derivatization process is discussed according to the analytical procedure used to achieve the reaction between the reagent and the target compounds (containing hydroxyl, thiol, amino, carbonyl and carboxyl as the main functional groups involved in derivatization). Important procedures for derivatization used in bioanalysis are in situ or based on extraction processes (liquid–liquid, solid‐phase and related techniques) applied to the biomatrix. In the review, chiral, isotope‐labeling, hydrophobicity‐tailored and post‐column derivatizations are also included, based on representative publications in the literature during the last two decades. Examples of derivatization reagents and brief reaction conditions are included, together with some bioanalytical applications and performances (chromatographic conditions, detection limit, stability and sample biomatrix).     

Determination of sulphonamides in animal tissues by high performance liquid chromatography with pre-column derivatization of 9-fluorenylmethyl chloroformate

A novel approach for simultaneous determination of 12 sulphonamides (sulphadiazine, sulphamethazine, sulphathiazole, sulphadimethoxine, sulphamerazine, sulphapyridine, sulphamethoxazole, suphamethizole, sulphaquinoxaline, sulphameter, sulphamonomethoxine, and sulphachloropyridazine) in animal tissues (swine muscle and liver, chicken muscle, beef muscle) by HPLC with UV detection has been developed. A pre-column derivatization of the sulphonamide compounds with 9-fluorenylmethyl chloroformate (FMOC-Cl) has been proposed and the reaction conditions have been optimized. The FMOC-sulphonamide derivatives were purified by SPE with silica gel as solid support prior to HPLC separation. The limits of detection for the sulphonamide compounds were greatly improved after the derivatization and purification step for the derivatives. Sulphonamide residues in animal tissues were extracted by acetonitrile and purified by solid phase extraction with C(18) as the solid support. The method developed has high sensitivity and good repeatability, and the average recoveries for most of the sulphonamides at various spiking levels were above 70% with relative standard deviations below 13.7%. The limits of detection for most sulphonamides can reach 3-5 microg/kg.     

Indirect fluorometric detection of tryptic digests separated by capillary zone electrophoresis

This work demonstrates the analytical utility of indirect fluorescence detection with capillary zone electrophoresis (CZE) for the analysis of trace quantities of macromolecular mixtures. Detection is based upon charge displacement and is not based upon any absorption or emission property of the analyte. No derivatization step is required. Indirect fluorescence is therefore a general detector for electrophoresis. Subfemtomolar quantities of tryptic digest mixtures are separated within three minutes, and reproducible peaks are obtained from the mixtures. Mass limits of detection are 3000 times lower than those of commercial high-performance liquid chromatography (HPLC) UV absorbance detectors and 180 times lower than those of UV absorbance detectors in CZE systems. This separation and detection system should be well suited to analysis of trace quantities of mixtures of peptides.     

Metal Cation Speciation via Extraction Reversed Phase HPLC with Refractive Index and/or Inductively Coupled Plasma Emission Detection Methods (HPLC-RI-ICP)

Abstract

Conventional high performance liquid chromatography (HPLC) instrumentation and packing materials can be inexpensively and rapidly utilized for the qualitative and quantitative analysis of various metal cations. The final approaches utilize reversed phase HPLC in the form of extraction chromatography. The detection of individually eluted, fully resolved metal cations is then possible via conventional refractive index (RI) and/or inductively coupled plasma ([icaron]P) emission spectroscopic detection. Final data presentation can be in the form of conventional, continuous RI or ICP chromatograms, via pulsed data ICP presentations, and/or via tabular ICP data presentation.     

Determination of methotrexate in environmental samples by solid phase extraction and high performance liquid chromatography: ultraviolet or tandem mass spectrometry detection?

In order to assess exposure levels of hospital personnel involved in the preparation and administration of antineoplastic drugs, environmental monitoring should be carried out. Wipe samples, pads, gloves and air samples should be collected at the end of each work shift, properly treated and then analysed using instrumental techniques which are sufficiently sensitive and specific to detect even trace amounts of drug. In this study, a method using high performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS), incorporating solid phase extraction (SPE), was validated for determination of methotrexate (MTX) in wipe and air samples. Each step of the method was first developed and optimised using ultraviolet detection (UV), and afterwards tandem mass spectrometry was used to obtain a lower limit of quantitation when the expected drug level was less than the analytical UV detection limit. SPE enabled a 20-fold preconcentration of the analyte when using HPLC/UV and a further 30-fold preconcentration was obtained when analysing samples by HPLC/MS/MS. For example, the limit of quantitation (LLQ) was lowered from 3000 ng on wipe (direct injection onto an HPLC/UV system) to 5 ng on wipe (SPE plus HPLC/MS/MS). 7-hydroxymethotrexate was used as internal standard to assess precision and accuracy.     

Second Derivative Ultraviolet Spectrophotometry and High Performance Liquid Chromatography with Fluorometric Detection of Cycloserine Using 9-Chloro-10-methyl Acridinium Triflate as a New UV and Fluorescent Labeling Agent

Abstract

Sensitive and simple second derivative UV spectrophotometric and HPLC with fluorometric detection methods were developed for cycloserine based on derivatization with 9-chloro-10-methyl acridinium triflate (CMAT) to yield a reaction product which absorbs in the UV at 361 nm and is fluorescent using excitation and emission wavelengths of 257 nm and 475 nm, respectively. The CMAT derivatization reaction takes 30 minutes at 70°C. Cycloserine was linear in the 0.3 – 5.0 μg/ml range (r=0.999, n=5) for the second derivative UV method and the 0.8 – 5 μg/ml range for the HPLC method (r=0.999, n=5). The limit of detection for cycloserine in the HPLC method can be improved to 0.15 μg/ml with the addition of glacial acetic acid to the analytical sample. The HPLC assay was applied to the determination of cycloserine in spiked human urine samples. The correlation coefficient (r) was in the 0.999 range and sensitivity was at the low pg/ml level.     

Selective quantification of ambroxol in human plasma by HPLC

The bronchosecretolytic drug ambroxol can be reliably quantified in human plasma by high performance liquid chromatography. Plasma is buffered alkaline, extracted with ether, and the organic solvent back-extracted with diluted acid. An automatically sampled aliquot is separated by reversed phase HPLC; the analyte is well separated from two metabolites that interfered strongly in earlier methods. UV detection at 230 nm enables a lower limit of quantitation of 5 ng/ml. Internal standardization with propranolol allows accurate and precise quantification. Evaluation of the optimized combination of mobile and stationary phase is described, and application of the method to experimental and clinical pharmacokinetic studies is illustrated.     

Development and validation of an HPLC method to quantify 3,4‐dideoxyglucosone‐3‐ene in peritoneal dialysis fluids

A method was developed and validated to quantify 3,4‐dideoxyglucosone‐3‐ene in peritoneal dialysis fluids by high‐performance liquid chromatography with UV detection after derivatization with o‐phenylenediamine. The advantages of this method compared with direct HPLC analysis are (i) the possibility of quantifying 3,4‐dideoxyglucosone‐3‐ene simultaneously together with other glucose degradation products, (ii) the compatibility of the method with MS detection for unequivocal identification of the analyte and (iii) a bathochromic shift of the UV absorbance maximum which leads to higher selectivity. The validated method was used to measure 3,4‐dideoxyglucosone‐3‐ene concentrations additionally to the glucose degradation products 3‐deoxyglucosone, methylglyoxal, glyoxal, 5‐hydroxymethylfurfural, 2‐furaldehyde, formaldehyde and acetaldehyde in 19 commercial products for peritoneal dialysis. Copyright © 2009 John Wiley & Sons, Ltd.     

Handling of environmental and biological samples via pre-column technologies

Sample handling is still a weak point in chromatography and in analytical chemistry in general. One consideration is the automation potential of new procedures. Solid-liquid extraction techniques in combination with pre-column technology are particularly promising in this regards. The construction and geometry of pre-columns both for conventional and narrow-bore HPLC are of major importance, since band broadening should be kept at a minimum for an optimal functioning of the analytical system. The various operations that can be carried out with such a pre-column are trace-enrichment, clean-up of the sample which depends on the type of adsorbents used in the precolumn, i.e., polar or apolar materials, ion exchangers or metal covered surfaces, etc., protection of the analytical column, field sampling and storage of samples and as a substrate for on-column chemical derivatizations. These various operations are demonstrated with practical examples from the fields of environmental and biological analysis. The selectivity can be further enhanced by coupling pre-column technology with selective detection models such as diode array UV, electrochemical or fluorescence detection. This enables the construction of optimal and integrated analysis systems which are fully automated and microprocessor controlled. They can also be made compatible with miniaturized LC-technology.